γ射线终末辐照对同种异体肌腱病毒灭活效果及生物力学性能影响的研究

目的研究γ射线终末辐照灭菌对同种异体肌腱病毒灭活的效果及对肌腱生物力学性能的影响。方法采用细胞培养法,检测γ射线辐照(照射剂量25 kGy)灭活PRV、VSV、PPV、EMCV、HIV-1病毒的效果。取正常供体手部肌腱12根,直径4~5 mm,随机分为实验组(辐照组)和对照组(新鲜不处理),进行病毒滴度检测,MTS力学试验机检测两组极限拉伸载荷。结果经过γ射线辐照可使污染在同种异体肌腱上的病毒滴度下降超过4 Log值,且盲传3代无病毒检出。辐照前后,同种异体肌腱极限拉伸载荷和组织结构差异无统计学意义(P0.05)。结论终末辐照灭菌(照射剂量25 kGy)是同种异体肌腱病毒灭活的有效方法,可避免引入其它化学试剂,且该工艺不会影响肌腱的极限拉伸载荷和组织结构。     

同种异体移植材料病毒灭活及其验证

异体组织移植是目前骨科临床上修复骨或软组织缺损、治疗骨折骨不连、重建肌腱韧带常用的方法.同种异体组织材料是以捐献的供体组织为原料经处理、加工或组成的产品.异体组织材料的临床应用首要问题是安全和有效.异体组织材料的有效性目前已经得到公认.异体骨移植能够替代自体骨进行骨移植,最终被受体骨爬行替代,实现骨的愈合;异体肌腱移植可以重建膝关节的交叉韧带及四肢肌腱;异体半月板移植可以起到自体半月板对膝关节的保护作用.安全性方面主要是疾病的传播,有研究证实,人免疫缺陷病毒(HIV-1)存在于HIV感染者的骨组织中,并能够通过骨移植传播.     

酒精保存同种异体骨治疗骨巨细胞瘤(附60例临床分析)

作者报告自1983年元月至1993年12月用酒精保存同种异体骨治疗骨巨细胞瘤60例，主张对肿瘤较小、骨壁完整者采用肿瘤彻底搔刮，75％酒精灭活，异体骨柴植人。对肿瘤较大、骨壁菲薄或不完整者应行大块植人或半关节置换以保留肢体功能。经2年以上随访引例，治愈率达87％。酒精灭活可杀死残存的肿瘤细胞，使细胞表面的蛋白质脱水而凝固。同时彻底刮净异体骨髓，减少术后反应，预防肿瘤复发。它具有制作简单，使用方便等优点。     

骨骼

NS398对人骨肉瘤细胞MG-63增殖与凋亡的影响,微波灭活骨再血管化的实验研究,电融法制备骨肉瘤融合细胞瘤苗的特性分析,大剂量甲氨蝶呤联合顺铂治疗骨肉瘤的临床研究,同种异体骨移植治疗般骨远端肿瘤的疗效评价,颈椎骨肿瘤的外科治疗分析,     

酒精保存同种异体骨治疗骨巨细胞瘤（附60例临床分析）

作者报告自１９８３年元月至１９９３年１２月用酒精保存同种异体骨治疗骨巨细胞瘤６０例，主张对种瘤产小，骨壁完整者采用肿瘤彻底搔刮，７５％酒精灭活，异体骨柴植入，对肿瘤较大，骨壁菲薄或不完整者应行大块植入或半关节置换以保留肢体功能，经２年以上随访５１例，治愈率达８７％，酒精灭活可杀死残存的肿瘤细胞，使细胞表面的蛋白质脱水而凝固，同时彻底刮净异体骨髓，减少术后反应，预防肿瘤复发，它具有制作简单，使用方便     

骨高转移人乳腺癌细胞株MDA-MB-231BM3及其动物模型的建立

目的:建立骨转移人乳腺癌细胞株MDA-MB-231BM3及其免疫缺陷小鼠骨转移动物模型.方法:将人乳腺癌细胞株MDA-MB-231改良后,接种于免疫缺陷小鼠左心室形成肿瘤骨转移,在放射性核素示踪下找到裸鼠骨转移病灶,然后切取病变骨组织进行体外培养,获得人乳腺癌骨转移细胞,用上述细胞重复以上体外-体内-体外循环3次,获得骨高转移人乳腺癌细胞株(MDA-MB-231BM3)及乳腺癌骨转移裸鼠模型.并将MDA-MB-231BM3与国外建株的人乳腺癌骨高转移细胞株MDA-MB-231BO在乳腺癌骨转移动物模型建立效率上进行比较.结果:获得以骨转移为主,兼以肺、肾上腺等组织转移的人乳腺癌细胞株MDA-MB-231BM3及其裸鼠骨转移动物模型.结论:MDA-MB-231BM3是以骨转移为主的高转移性人乳腺癌细胞株,该细胞株及其骨转移动物模型为乳腺癌转移的生物学研究提供了一个良好的技术平台.     

高滴度逆转录病毒载体4种转染方法对小鼠脾脏T细胞转染率的比较

逆转录病毒为研究和应用最广泛的一种病毒载体 ,应用衣霉素 (Ｔｕｎｉｃａｍｙｃｉｎ)处理包装细胞后病毒滴度明显提高 ,在此基础上应用 4种方法转染小鼠脾脏Ｔ细胞 ,比较4种方法的转染率。脂质体法转染包装细胞ＧＰ Ｅ86 ,Ｇ4 1 8筛选抗性克隆 ,收集病毒上清。为提高病毒滴度 ,我们采用一种新方法 :ＧＰ Ｅ86细胞转染前先在含ｔｕｎｉｃａｍｙｃｉｎ 1 2 0ｎｇ/ｍｌ的培养液中培养 1 8ｈ ,再用新鲜病毒上清转染 ,筛选过程同上。ＮＩＨ3Ｔ3细胞测定病毒滴度约 5× 1 0 6ｃｆｕ/ｍｌ。 2 0 ｇ左右的ＢＬＡＢ/ｃ小鼠脱颈处死 ,将脾脏制成单…     

放射性核素骨显像在建立人肺腺癌骨转移细胞株中的应用

目的:探讨采用放射性核素骨显像技术建立人肺腺癌骨转移细胞株SIC-A-1BM以及在免疫缺陷BNX小鼠的转移动物模型活体成像中的作用.方法:将人肺腺癌细胞株SIC-A-1注射入小鼠左心室,形成骨转移,在放射性核素骨显像剂示踪下寻找到骨转移灶,然后切除病变组织进行体外培养以获得转移性肺腺癌细胞.利用获得的第1代肺腺癌骨转移细胞,经血液(动、静脉系统)和肺原位途径进行种植,按上述步骤重复多个循环.结果:染色体分析结果确定亲代SPC-A-1细胞经小鼠体内-体外连续筛选,通过放射性核素骨显像后显示,获得的肺腺癌骨转移细胞株(SIC-A-1BM)未改变人源细胞属性.99mTcMDP和X射线的放射剂量对SPC-A-1BM细胞生长影响的结果表明,111 MBq的99mTc-MDP对人肺腺癌骨转移细胞生长的影响类似于且略小于40 kV、2 mA、4 s的X射线.放射性核素骨显像与X线摄片检测出的小鼠骨转移的敏感度、特异度和准确度分别为97.6%和31.3%、73.3%和100%以及94%和43%.结论:放射性核素骨显像技术为建立人肺腺癌骨转移细胞株SIC-A-1BM及其免疫缺陷BNX小鼠转移动物模型活体成像提供了一种实用且便捷的实验手段.     

重组腺病毒人内皮抑素的质量标准研究

目的: 建立重组腺病毒人内皮抑素的质量标准和检测方法. 方法: PCR法鉴定腺病毒载体的E2B区和插入基因,琼脂糖凝胶电泳检查重组病毒DNA酶切图谱.分光光度法测定病毒颗粒数,TCID50法测定病毒感染滴度.感染人肝癌细胞HepG2测定插入基因的表达量,感染人血管内皮细胞HM2测定表达产物的生物学活性.病毒的纯度分析采用A260/A280比值和HPLC法进行.采用A549细胞进行复制型腺病毒的检测.结果: 腺病毒载体E2B区和插入基因PCR扩增结果与理论相符,重组病毒DNA的酶切图谱与标准品一致.原液病毒颗粒数为2.4×1012 VP/ml,滴度为1.53×1011 IU/ml,比滴度为6.4% IU/VP;成品病毒颗粒数为1.0×1012 VP/ml,滴度为3.75×1010 IU/ml,比滴度为病3.8% IU/VP.以50 MOI重组腺病毒感染HepG2细胞48 h后培养上清人内皮抑素表达量为332 ng/ml.50 MOI重组腺病毒对血管内皮细胞生长的抑制率为55%.A260/A280为1.29,HPLC纯度为99.7%.复制型腺病毒为≤1RCA/3×1010 VP.其他各项检测指标均符合规定.结论: 建立了重组腺病毒人内皮抑素的质量标准及其检测方法,并用于该产品的质量控制.     

同种异体脱钙骨移植修复下颌骨缺损的临床研究

目的 应用同种异体脱钙骨移植,修复因肿瘤／瘤样病变切除后的下颌骨缺损,并研究其临床适应征、方法和要求。方法 异体脱下下颌骨按Urist氏法制备成部分脱钙骨,术中即刻植入,截骨端采用钛板或钢丝结扎固定。结果 16例中14例获得成功,宿主接受脱钙骨移植术后3－5天,术区明显肿胀、渗出、体温达39℃,7－10天恢复正常。术后3－6月X线摄片移植骨与宿主骨界面模糊,有骨痂形成。随访颌面外形、下颌运动、咬He关系,张口度恢复正常。结论 经脱钙处理的异体下颌骨免疫反应轻,形态自然,成形方便,有较好的机械支撑强度和成骨诱导能力,是修复下颌骨缺损的理想替代材料。     

HIV-1 infectivity of human carcinoma cell lines lacking CD4 receptors.

Human immunodeficiency virus type 1 (HIV-1) prototype, HIV1 LAV, and a Zairian virus HIV1 NDK, an isolate highly cytopathic for CD4+ lymphocytes, were used to infect eleven different CD4 negative non-lymphoid human cell lines. Eight of the lines were derived from carcinomas wherein human papillomavirus was thought to have been etiologic. All these cell lines lacked CD4 receptor and CD4 specific mRNA. After cocultivation with sensitive CEM cells, HIV-1 LAV was rescued from six infected cell lines and HIV-1 NDK from nine. Shedding of free virus into the culture medium was observed in three cell lines infected by HIV-1 NDK and in only one cell line infected by HIV-1 LAV. The infectibility of CD4 negative cell lines indicates that both HIV-1 strains were able to use a CD4 independent mechanism to infect the cells; however, HIV-1 NDK showed the higher efficiency of infection. This virus was also able to overcome the intracellular block of viral reproduction. These results suggest that a broader spectrum of cell types of non-lymphoid origin lacking the CD4 receptor can serve as a viral reservoir. In some cases they are direct producers of infectious HIV-1 particles. This suggests, that in addition to immunosuppressive mechanisms, HIV-1 could play a more direct role in induction of neoplastic changes.     

Induction of Epstein-Barr virus in B-lymphoblastoid cells by human immunodeficiency virus type 1

Individuals infected with the human immunodeficiency virus (HIV), the etiologic agent of acquired immunodeficiency syndrome (AIDS), often show symptoms associated with reactivation of Epstein-Barr virus (EBV). In this study, we show that exposure of EBV-positive B lymphocytes to HIV-1 in vitro induced the EBV replicative cycle in these cells, as evidenced by an increased proportion of cells expressing EBV early antigens (EA) and capsid antigens (VCA). Reactivation of EBV by HIV-1 appeared to be virus-dose-dependent and required virus penetration and expression in B cells. Although HIV-1 RNA was detected by in situ hybridization in the majority of HIV-1-infected B lymphocytes, induction of EA and VCA was transient and limited to less than 20% of the cell population. The tumor-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and HIV-1 acted synergistically and had similar kinetics in inducing the expression of EBV. Direct reactivation of EBV by HIV-1 may contribute to the role of EBV as a factor in the genesis of AIDS-related conditions.     

EB病毒相关的人类免疫缺陷病毒阴性浆母细胞淋巴瘤的临床病理分析

目的 探讨人类免疫缺陷病毒（HIV）阴性且无免疫缺陷的浆母细胞淋巴瘤（PBL）的临床病理特征,提高对这组疾患的认识.方法 回顾性分析6例无免疫缺陷且HIV-PBL的组织学特点,原位杂交染色检测EB病毒（EBV）感染状态.分别采用免疫组织化学SP法及荧光原位杂交（FISH）技术检测PBL的免疫表型、EBV潜伏类型,探索myc基因的易位.结果 HIV-PBL表现为浆母细胞样或免疫母细胞样细胞的单一增生,可见瘤巨细胞及坏死;背景反应细胞少,核分裂象较多.所有病例都有EBV感染,潜伏类型为Ⅰ型（LMP1^-及EBNA2^-）.肿瘤细胞表达B细胞终末分化阶段的表型CD20^-/CD3^-/CD1386＋/Kappa＋或Lambda^＋.6例HIV-PBL均为老年患者（中位年龄69.5岁）,男女各3例;结外及口腔外侵犯率高,分别为6、5例.中位生存期为25.5个月.此外,3例患者具有免疫球蛋白重链（IgH）与myc基因易位.结论 HIV-PBL是一组独立疾患,具有无HIV感染、老年人、EBV阳性、结外及口腔外侵犯率高等特点,应与HIV＋的PBL相区别.     

Doxorubicin stimulates transcription from the human immunodeficiency virus long terminal repeat sequences.

A recombinant plasmid carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1) linked to the reporter chloramphenicol acetyl transferase (CAT) gene was stably introduced into rat liver cells. The transfectant cells expressed CAT activity from the HIV LTR. The response to doxorubicin was studied and it was found that at the optimum concentration of 20 micrograms/ml doxorubicin, the expression of CAT from the HIV LTR was stimulated by 65-fold. Our results suggest caution against therapy including doxorubicin in the treatment of AIDS patients.     

Inactivation of HIV-1 and HIV-2 by various manufacturing procedures for human plasma proteins

Human retroviruses causing AIDS (HIV-1, HIV-2) can occur in human plasma donations. Since HIV-contaminated plasma cannot be completely excluded by testing for anti-HIV-1 (routine plasma screening for anti-HIV-2 has not yet been established), a safeguard against AIDS in therapeutics derived from human plasma can only be achieved by introducing HIV inactivating/eliminating methods into the manufacturing process of plasma derivatives. To investigate the HIV inactivating efficiency of such methods, aliquots of infectious HIV-1 or HIV-2 concentrates were added to a protein preparation, the resulting HIV spiked preparation was then treated according to the method to be studied, and the amount of infectious HIV in this preparation was determined before and after treatment. Methods by which HIV-1 or HIV-2, respectively, were completely inactivated were ethanol fractionation according to the Cohn procedure, pepsin treatment, affinity chromatography, protein precipitation by various methods, and pasteurization (heat treatment at 60 degrees C in aqueous solution). The use of these methods for manufacturing human plasma derivatives resulted in products that were free of any infectious HIV-1 or HIV-2 and thus unable to transmit AIDS.     

Species tropism of HIV-1 infectivity of interspecific cell hybridomas implies non-CD4 structures are required for cell entry

Studies have demonstrated that CD4 may be necessary but not sufficient for human immunodeficiency virus (HIV) infection. Murine cell lines constructed to express the human CD4 glycoprotein demonstrate a restriction in their ability to support infection by the HIV-1 retrovirus. Such restrictions may indicate that molecules in addition to CD4 may be necessary for HIV-1 infection. HIV-1 infectivity has been examined in a panel of murine and human cell lines. It has been demonstrated that CD4 expression allows infectivity on a wide range of human cell lines. Some interspecific hybridomas constructed between human and murine cell lines can, when expressing the human CD4 glycoprotein, become infectable in a manner similar to human cell lines. These studies imply that surface molecules, in addition to CD4, are necessary for HIV-1 entry and infectivity. Such molecules will be important for understanding the pathogenesis of HIV-1.     

Selective killing of human immunodeficiency virus-infected and -producing cells by liposomes containing diphtheria toxin fragment A

Liposomes containing fragment A of diphtheria toxin killed human immunodeficiency virus (HIV)-producing MOLT-4 or TALL-1 cells, and HTLV-I-carrying MT-4 cells infected with HIV, but did not kill these cells when they were not infected with HIV. This killing was not affected by the presence of HIV antibodies. These results show that liposomes containing fragment A of diphtheria toxin selectively killed cells expressing HIV antigens or producing the virus in vitro.     

Impaired in-vitro proliferation of hemopoietic precursors in HIV-1-infected subjects

Patients with acquired immunodeficiency syndrome (AIDS) and persistent lymphadenopathy syndrome (LAS) display significant hematological abnormalities of one or more cell lineages. In order to understand the pathophysiologic mechanisms leading to these abnormalities we studied the proliferation capacity of pluripotent and committed hemopoietic precursors using in-vitro colony assays. Anemia, leukopenia and thrombopenia were relatively frequent findings in HIV-infected subjects irrespectively of the patients' clinical status. The colony growth capacity of AIDS patients' GM-CFU and BFU-E was significantly decreased whereas no GEMM-CFU colonies could be obtained. There was no correlation between the number of BFU-E and GM-CFU colony number and the hemoglobin or the absolute number of polynuclear cells, respectively. The plating efficiency of both committed and pluripotent hematopoietic precursors from HIV infected patients could not be enhanced when additional exogenous recombinant GM-CSF, human interleukin 3 or erythropoietin were added in contrast to normal patients' cells. In addition, the impaired colony growth of these precursors could not be restored after adherent or T-cell depletion or the addition of normal allogenic irradiated adherent or/and T cells. Since this colony growth abnormality was also detected in HIV seropositive asymptomatic subjects our findings strongly suggest that the in-vitro growth of hematopoietic precursors is affected early after HIV-1 infection.     

Human immunodeficiency virus type 1 tat gene up-regulates interleukin 4 receptors on a human B-lymphoblastoid cell line.

The human immunodeficiency virus type I (HIV-1) regulatory gene, tat III, is a powerful trans-activator of gene expression from the viral long terminal repeat and is essential for HIV replication. In addition, tat III protein has been shown to be immunosuppressive as indicated by the inhibition of antigen mediated T-cell proliferation. To further test whether tat III might play a direct role in the immunosuppressive effects of HIV-1 in addition to its role in virus replication, we examined the regulation of interleukin 4 (IL-4) receptors on a human B-lymphoblastoid cell line (Raji) transfected with HIV-1 tat gene (Raji-tat III). We used radioligand receptor binding analysis for cell surface expression and Northern blot analysis for the expression of human IL-4 receptor gene in Raji-tat III cells. Control Raji cells expressed 1383 +/- 361 (SE; n = 3) IL-4 binding sites/cell with a dissociation constant (Kd) of 144 +/- 27 pM (n = 3). However, Raji-tat III cells expressed about three times higher IL-4 receptors (4000 +/- 633 IL-4 binding sites/cell; P less than 0.03 compared to Raji cells) with a similar Kd of 273 +/- 90 pM (n = 3; P greater than 0.05 compared to Raji cells). Whereas both Raji and Raji-tat III cells exhibited a single mRNA species (approximately 4 kilobases) of IL-4 receptors by Northern blot analysis, the mRNA level was about 3-fold higher in Raji-tat III cells compared to Raji cells. Cycloheximide inhibited the expression of IL-4 receptors by 50% in about 2 h in both cell types indicating both the half-life of IL-4 receptors and the requirement for protein synthesis for the tat III up-regulation of IL-4 receptors. Since IL-4 under certain circumstances has been shown to be immunosuppressant, our observation that the HIV-1 tat gene up-regulates IL-4 receptors suggests the possibility that the immunosuppressive effects of HIV-1 are mediated at least in part through IL-4 receptors.